記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Scientific Reports  

Neuronal ceroid lipofuscinosis is one of many neurodegenerative storage diseases characterized by excessive accumulation of lipofuscins. CLN10 disease, an early infantile neuronal ceroid lipofuscinosis, is associated with a gene that encodes cathepsin D (CtsD), one of the major lysosomal proteases. Whole body CtsD-knockout mice show neurodegenerative phenotypes with the accumulation of lipofuscins in the brain and also show defects in other tissues including intestinal necrosis. To clarify the precise role of CtsD in the central nervous system (CNS), we generated a CNS-specific CtsD-knockout mouse (CtsD-CKO). CtsD-CKO mice were born normally but developed seizures and their growth stunted at around postnatal day 23 ± 1. CtsD-CKO did not exhibit apparent intestinal symptoms as those observed in whole body knockout. Histologically, autofluorescent materials were detected in several areas of the CtsD-CKO mouse’s brain, including: thalamus, cerebral cortex, hippocampus, and cerebellum. Expression of ubiquitin and autophagy-associated proteins was also increased, suggesting that the autophagy-lysosome system was impaired. Microglia and astrocytes were activated in the CtsD-CKO thalamus, and inducible nitric oxide synthase (iNOS), an inflammation marker, was increased in the microglia. Interestingly, deposits of proteinopathy-related proteins, phosphorylated α-synuclein, and Tau protein were also increased in the thalamus of CtsD-CKO infant mice. Considering these results, we propose thatt the CtsD-CKO mouse is a useful mouse model to investigate the contribution of cathepsin D to the early phases of neurodegenerative diseases in relation to lipofuscins, proteinopathy-related proteins and activation of microglia and astrocytes.

DOI： 10.1038/s41598-022-15805-3

Scopus

PubMed

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biomedical Research (Japan)  

A live assay tool has been established to uncover the precise molecular mechanisms underlying complex cell fusion events in myoblasts. The novel cell-based assay, HiMy (HiBiT-based myo-blast fusion), utilizes a recently developed split-luciferase technology. The assay successfully detected cell fusion in differentiating C2C12 myoblast cultures. This allowed us to measure mixing of the cytoplasm, which occurred several hours after the initiation of C2C12 differentiation. Un-like what was reported earlier, the fusion was detected a few hours after the initiation of differen-tiation. Thus, this assay is sensitive enough to monitor fusion events before they become detectable using conventional methods. Furthermore, a panel of laboratory compounds, including a variety of inhibitors of cellular enzymes or activities, were assayed using the HiMy assay. Lovas-tatin, a cholesterol biogenesis inhibitor, decreased HiMy activity by approximately 50%. In con-trast, mevalonolactone, a precursor for cholesterol synthesis, increased fusion activity. These results confirmed the previous finding that the amount of cellular cholesterol positively correlates with the rate of myoblast fusion during myogenesis. These results indicate that the novel cell fusion assay is a quick, accurate, and robust method to monitor intercellular fusion events.

DOI： 10.2220/biomedres.43.107

Scopus

記述言語：日本語   出版者・発行元：Frontiers in Neuroanatomy  

Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography (i.e., serial section SEM). This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods.

DOI： 10.3389/fnana.2021.759804

Scopus

DOI： 10.1002/glia.23843

Scopus

PubMed

その他リンク： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/glia.23843

出版者・発行元：公益社団法人 日本顕微鏡学会  

<p>切片SEM法は，新たな走査電子顕微鏡（SEM）イメージング技法であり，スライドガラスに貼り付けた樹脂包埋組織切片から反射電子（BSE）像を観察する手法である．この手法では，高度なミクロトーム技術を必要とすることなく，透過電子顕微鏡のような超薄切片像を取得することができる．そこで本稿では，私たちが独自に開発した3つの切片SEM法，1）準超薄切片から超薄像をイメージする手法（準超薄切片SEM法），2）蛍光像と準超薄切片BSE像の相関顕微鏡観察法（CLEM法），3）凍結薄切法（徳安法）との組み合わせ法，について紹介する．さらに，新たな3Dイメージング技法である「連続切片SEM法」についても紹介し，切片のSEMイメージング技術の可能性について考察する．</p>

DOI： 10.11410/kenbikyo.55.1_18

出版者・発行元：(公社)日本顕微鏡学会  

切片SEM法は、新たな走査電子顕微鏡(SEM)イメージング技法であり、スライドガラスに貼り付けた樹脂包埋組織切片から反射電子(BSE)像を観察する手法である。この手法では、高度なミクロトーム技術を必要とすることなく、透過電子顕微鏡のような超薄切片像を取得することができる。そこで本稿では、私たちが独自に開発した3つの切片SEM法、1)準超薄切片から超薄像をイメージする手法(準超薄切片SEM法)、2)蛍光像と準超薄切片BSE像の相関顕微鏡観察法(CLEM法)、3)凍結薄切法(徳安法)との組み合わせ法、について紹介する。さらに、新たな3Dイメージング技法である「連続切片SEM法」についても紹介し、切片のSEMイメージング技術の可能性について考察する。(著者抄録)